Chemokines are multifunctional mediators responsible for recruitment and activation of the leukocyte at the site of inflammation. They play key roles in many acute and chronic inflammatory diseases including rheumatoid arthritis and asthma, and mediate tumor cell trafficking and metastasis. Chemokine functions are mediated via specific G-protein coupled receptors (GPCRs). Upon activation, these receptors are desensitized and down-regulated. Chemokines and chemokine receptors are redundant in their interaction. A long-term goal of this laboratory is to delineate the molecular mechanisms via which chemokines trigger selective inflammatory responses and receptor regulation. Works supported by this grant over the past 11 years have shown that these receptors are capable of cross-regulating each other's functions thereby, limiting cellular responsiveness to chemokines. The Interleukin-8 (IL-8/CXCL8) receptors (CXCR1 and CXCR2) bind CXCL8 with similar affinity to initiate leukocyte chemotaxis, but only CXCR1 mediates respiratory burst and cross-regulatory signals. Preliminary studies have identified the G protein-coupled receptor kinase 6 (GRK6), and the activator of G protein signaling 3 (AGS3) as important regulators of CXCR2-mediated leukocyte functions. The hypothesis that underlies this proposal is that CXCR2 couples to GRK6 and AGS3 to regulate cellular responses to CXCL8. The overall objective is to define the molecular mechanisms via which GRK6 and AGS3 modulate CXCR2 activation and regulation. The specific aims are to determine 1) the role of GRK6 in CXCR2- mediated leukocyte activation and regulation, 2) the role of GRK6 and AGS3 interaction in modulating CXCR2 functions and, 3) the role of GRK6 and AGS3 in CXCR2-mediated post-endocytic signals. The proposed experiments will use a multiplicity of approaches including leukocytes, RBL-2H3 cells stably expressing wild type and mutants CXCR1, CXCR2, AGS3 and GRK6, as well as mice deficient in GRK6, AGS3, (arr2, CXCR1 and CXCR2. The understanding of the molecular mechanisms of CXCL8 receptors activation and regulation will aid in understanding the control of inflammation as well as the etiology of many inflammatory disorders. Such information will provide new targets and new assays for the development of therapeutics which modulate inflammation, wound healing and tumor progression. PUBLIC HEALTH RELEVANCE: Chemokines are involved in many acute and chronic inflammatory diseases including rheumatoid arthritis (RA), multiple sclerosis, asthma, cystic fibrosis, chronic bronchitis and bronchiectasis and HIV infection and progression. The redundancy in receptor activation, however, makes it difficult to target chemokines for therapeutic intervention. CXCL8 interacts with two receptors, CXCR1 and CXCR2. CXCR1 is specific for CXCL8 whereas CXCR2 binds six different CXC chemokines to activate leukocyte functions. Upon activation, CXCR2 scaffolds with several regulatory proteins (i.e. G protein coupled receptor specific kinase 6 (GRK6), activator of G protein signaling 3, (AGS3) and beta-arrestin 2 ((arr2) to modulate cellular responses. The studies described in this application, will define the importance of GRK6, AGS3 and 2arr2 in CXCR2-mediated cellular responses. Such information will aid in understanding the control of inflammation as well as the etiology of many inflammatory disorders. These studies will also provide new targets for the development of therapeutics to modulate inflammation.